Who benefits from granulomas, mycobacteria or host?

By investigating host-pathogen interactions in zebrafish using intravital imaging, Davis and Ramakrishnan (2009) provide evidence that aggregates of immune cells known as granulomas, long thought to constrain mycobacterial infection, may instead facilitate its spread.

Single cell resolution of SARS-CoV-2 tropism, antiviral responses, and susceptibility to therapies in primary human airway epithelium.

The human airway epithelium is the initial site of SARS-CoV-2 infection. We used flow cytometry and single cell RNA-sequencing to understand how the heterogeneity of this diverse cell population contributes to elements of viral tropism and pathogenesis, antiviral immunity, and treatment response to remdesivir. We found that, while a variety of epithelial cell types are susceptible to infection, ciliated cells are the predominant cell target of SARS-CoV-2. The host protease TMPRSS2 was required for infection of these cells. Importantly, remdesivir treatment effectively inhibited viral replication across cell types, and blunted hyperinflammatory responses. Induction of interferon responses within infected cells was rare and there was significant heterogeneity in the antiviral gene signatures, varying with the burden of infection in each cell. We also found that heavily infected secretory cells expressed abundant IL-6, a potential mediator of COVID-19 pathogenesis.

Cutting Edge: Nucleocapsid Vaccine Elicits Spike-Independent SARS-CoV-2 Protective Immunity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Neutralizing Abs target the receptor binding domain of the spike (S) protein, a focus of successful vaccine efforts. Concerns have arisen that S-specific vaccine immunity may fail to neutralize emerging variants. We show that vaccination with a human adenovirus type 5 vector expressing the SARS-CoV-2 nucleocapsid (N) protein can establish protective immunity, defined by reduced weight loss and viral load, in both Syrian hamsters and K18-hACE2 mice. Challenge of vaccinated mice was associated with rapid N-specific T cell recall responses in the respiratory mucosa. This study supports the rationale for including additional viral Ags in SARS-CoV-2 vaccines, even if they are not a target of neutralizing Abs, to broaden epitope coverage and immune effector mechanisms.

Cutting Edge: Mouse SARS-CoV-2 Epitope Reveals Infection and Vaccine-Elicited CD8 T Cell Responses.

The magnitude of SARS-CoV-2-specific T cell responses correlates inversely with human disease severity, suggesting T cell involvement in primary control. Whereas many COVID-19 vaccines focus on establishing humoral immunity to viral spike protein, vaccine-elicited T cell immunity may bolster durable protection or cross-reactivity with viral variants. To better enable mechanistic and vaccination studies in mice, we identified a dominant CD8 T cell SARS-CoV-2 nucleoprotein epitope. Infection of human ACE2 transgenic mice with SARS-CoV-2 elicited robust responses to H2-Db/N219-227, and 40% of HLA-A*02+ COVID-19 PBMC samples isolated from hospitalized patients responded to this peptide in culture. In mice, i.m. prime-boost nucleoprotein vaccination with heterologous vectors favored systemic CD8 T cell responses, whereas intranasal boosting favored respiratory immunity. In contrast, a single i.v. immunization with recombinant adenovirus established robust CD8 T cell memory both systemically and in the respiratory mucosa.

Immune Profiling to Determine Early Disease Trajectories Associated With Coronavirus Disease 2019 Mortality Rate: A Substudy from the ACTT-1 Trial.

Coronavirus disease 2019 (COVID-19) outcomes are linked to host immune responses and may be affected by antiviral therapy. We investigated antibody and cytokine responses in ACTT-1 study participants enrolled at our center. We studied serum specimens from 19 hospitalized adults with COVID-19 randomized to treatment with remdesivir or placebo. We assessed severe acute respiratory syndrome coronavirus 2 antibody responses and identified cytokine signatures, using hierarchical clustering. We identified no clear immunologic trends attributable to remdesivir treatment. Seven participants were initially seronegative at study enrollment, and all 4 deaths occurred in this group with more recent symptom onset. We identified 3 dominant cytokine signatures, demonstrating different disease trajectories.

Cerebrospinal Fluid Bacillary Load by Xpert MTB/RIF Ultra Polymerase Chain Reaction Cycle Threshold Value Predicts 2-Week Mortality in Human Immunodeficiency Virus-Associated Tuberculous Meningitis.

BACKGROUND: The World Health Organization recommends GeneXpert MTB/RIF Ultra (Xpert Ultra), a fully automated polymerase chain reaction (PCR) assay, as the initial tuberculous meningitis (TBM) diagnostic test. The assay's PCR cycle threshold (Ct) values represent the number of PCR cycles required for probe signal to be detected (low Ct value = high bacillary load) and may approximate tuberculosis (TB) bacillary load. We measured the relationship between cerebrospinal fluid (CSF) TB bacillary load with mortality. METHODS: We prospectively enrolled 102 human immunodeficiency virus (HIV)-positive Ugandans with probable or definite TBM from April 2015 to August 2019. Xpert Ultra Ct tertiles and semi-quantitative categories were separately analyzed as predictors of 2-week mortality. We investigated associations between Ct and baseline clinical and CSF parameters. RESULTS: Subjects with Ct values in the low tertile (ie, high bacillary load) had 57% 2-week mortality-worse than the intermediate (17%) and high (25%) Ct tertiles and Xpert Ultra-negative (30%) probable TBM cases (P = .01). In contrast, the reported semi-quantitative Xpert Ultra categorization was less precise; with the medium to low category trending toward worse 2-week survival (42%) compared with very low (28%), trace (26%), and negative (30%) categories (P = .48). Ct tertile was significantly associated with baseline CSF lactate (P = .03). CONCLUSIONS: High CSF TB bacillary load, as measured by Xpert Ultra Ct tertile, is associated with an almost 2-fold higher 2-week mortality in HIV-associated TBM and is a better predictor than the reported Xpert Ultra semi-quantitative category. Xpert Ultra Ct values could identify TBM patients at increased risk of death who may benefit from enhanced supportive care.

SARS-CoV-2 neutralization and serology testing of COVID-19 convalescent plasma from donors with nonsevere disease.

BACKGROUND: The transfer of passive immunity with convalescent plasma is a promising strategy for treatment and prevention of COVID-19, but donors with a history of nonsevere disease are serologically heterogenous. The relationship between SARS-Cov-2 antigen-binding activity and neutralization activity in this population of donors has not been defined. STUDY DESIGN AND METHODS: Convalescent plasma units from 47 individuals with a history of nonsevere COVID-19 were assessed for antigen-binding activity of using three clinical diagnostic serology assays (Beckman, DiaSorin, and Roche) with different SARS-CoV-2 targets. These results were compared with functional neutralization activity using a fluorescent reporter strain of SARS-CoV-2 in a microwell assay. RESULTS: Positive correlations of varying strength (Spearman r = 0.37-0.52) between antigen binding and viral neutralization were identified. Donors age 48 to 75 years had the highest neutralization activity. Units in the highest tertile of binding activity for each assay were enriched (75%-82%) for those with the highest levels of neutralization. CONCLUSION: The strength of the relationship between antigen-binding activity and neutralization varies depending on the clinical assay used. Units in the highest tertile of binding activity for each assay are predominantly comprised of those with the greatest neutralization activity.

A eulogy for Dr Francisco Miguel Marty Forero.

As some of those who were lucky enough to have been mentored by Dr Francisco Marty in transplant infectious diseases, we stand with the larger medical community in mourning his untimely death and in commemorating him as a uniquely exceptional and talented physician, investigator, teacher, mentor, friend, artist, and human being.

Use of triazoles for the treatment of invasive aspergillosis: A three-year cohort analysis.

In a 3-year cohort study of adult patients with proven or probable IA, fewer patients initially treated with isavuconazole experienced adverse events compared with voriconazole, but more patients required a change in therapy due to lack of clinical efficacy.

Risk of Latent Tuberculosis Reactivation After Hematopoietic cell Transplantation.

There were no cases of tuberculosis in a cohort of 2531 patients who underwent hematopoietic cell transplantation from 2010 to 2015 after 7323 person-years of follow up (95% confidence interval [CI], 0.0-0.05 cases/100 person-years), including 29 (1.15%) patients with untreated latent tuberculosis after 89 person-years of follow-up (95% CI, 0.0-4.06 cases/100 person-years).

Rapid Detection of Powassan Virus in a Patient With Encephalitis by Metagenomic Sequencing.

We describe a patient with severe and progressive encephalitis of unknown etiology. We performed rapid metagenomic sequencing from cerebrospinal fluid and identified Powassan virus, an emerging tick-borne flavivirus that has been increasingly detected in the United States.

CD4+ T cell-dependent IFN-γ production by CD8+ effector T cells in Mycobacterium tuberculosis infection.

Both CD4+ and CD8+ T cells contribute to immunity to tuberculosis, and both can produce the essential effector cytokine IFN-γ. However, the precise role and relative contribution of each cell type to in vivo IFN-γ production are incompletely understood. To identify and quantitate the cells that produce IFN-γ at the site of Mycobacterium tuberculosis infection in mice, we used direct intracellular cytokine staining ex vivo without restimulation. We found that CD4+ and CD8+ cells were predominantly responsible for production of this cytokine in vivo, and we observed a remarkable linear correlation between the fraction of CD4+ cells and the fraction of CD8+ cells producing IFN-γ in the lungs. In the absence of CD4+ cells, a reduced fraction of CD8+ cells was actively producing IFN-γ in vivo, suggesting that CD4+ effector cells are continually required for optimal IFN-γ production by CD8+ effector cells. Accordingly, when infected mice were treated i.v. with an MHC-II-restricted M. tuberculosis epitope peptide to stimulate CD4+ cells in vivo, we observed rapid activation of both CD4+ and CD8+ cells in the lungs. Indirect activation of CD8+ cells was dependent on the presence of CD4+ cells but independent of IFN-g responsiveness of the CD8+ cells. These data provide evidence that CD4+ cell deficiency impairs IFN-γ production by CD8+ effector cells and that ongoing cross-talk between distinct effector T cell types in the lungs may contribute to a protective immune response against M. tuberculosis. Conversely, defects in these interactions may contribute to susceptibility to tuberculosis and other infections.

CD4 + T cells recruit, then engage macrophages in cognate interactions to clear Mycobacterium tuberculosis from the lungs.

IFN-γ-producing CD4 + T cells are required for protection against lethal Mycobacterium tuberculosis ( Mtb ) infections. However, the ability of CD4 + T cells to suppress Mtb growth cannot be fully explained by IFN-γ or other known T cell products. In this study, we show that CD4 + T cell-derived IFN-γ promoted the recruitment of monocyte-derived macrophages (MDMs) to the lungs of Mtb -infected mice. Although the recruited MDMs became quickly and preferentially infected with Mtb , CD4 + T cells rapidly disinfected the MDMs. Clearance of Mtb from MDMs was not explained by IFN-γ, but rather by MHCII-mediated cognate interactions with CD4 + T cells. These interactions promoted MDM expression of glycolysis genes essential for Mtb control. Thus, by recruiting MDMs, CD4 + T cells initiate a cycle of bacterial phagocytosis, Mtb antigen presentation and disinfection in an attempt to clear the bacteria from the lungs.

Adrenal gland macrophages regulate glucocorticoid production through Trem2 and TGF-ß.

Glucocorticoid synthesis by adrenal glands (AGs) is regulated by the hypothalamic-pituitary-adrenal axis to facilitate stress responses when the host is exposed to stimuli. Recent studies implicate macrophages as potential steroidogenic regulators, but the molecular mechanisms by which AG macrophages exert such influence remain unclear. In this study, we investigated the role of AG macrophages in response to cold challenge or atherosclerotic inflammation as physiologic models of acute or chronic stress. Using single-cell RNA sequencing, we observed dynamic AG macrophage polarization toward classical activation and lipid-associated phenotypes following acute or chronic stimulation. Among transcriptional alterations induced in macrophages, triggering receptor expressed on myeloid cells 2 (Trem2) was highlighted because of its upregulation following stress. Conditional deletion of macrophage Trem2 revealed a protective role in stress responses. Mechanistically, Trem2 deletion led to increased AG macrophage death, abolished the TGF-ß-producing capacity of AG macrophages, and resulted in enhanced glucocorticoid production. In addition, enhanced glucocorticoid production was replicated by blockade of TGF-ß signaling. Together, these observations suggest that AG macrophages restrict steroidogenesis through Trem2 and TGF-ß, which opens potential avenues for immunotherapeutic interventions to resolve stress-related disorders.

SARS-CoV-2 infection is associated with intestinal permeability, systemic inflammation, and microbial dysbiosis in hospitalized patients.

Coronavirus disease 2019 (COVID-19) and its associated severity have been linked to uncontrolled inflammation and may be associated with changes in the microbiome of mucosal sites including the gastrointestinal tract and oral cavity. These sites play an important role in host-microbe homeostasis, and disruption of epithelial barrier integrity during COVID-19 may potentially lead to exacerbated inflammation and immune dysfunction. Outcomes in COVID-19 are highly disparate, ranging from asymptomatic to fatal, and the impact of microbial dysbiosis on disease severity is unclear. Here, we obtained plasma, rectal swabs, oropharyngeal swabs, and nasal swabs from 86 patients hospitalized with COVID-19 and 12 healthy volunteers. We performed 16S rRNA sequencing to characterize the microbial communities in the mucosal swabs and measured concentrations of circulating cytokines, markers of gut barrier integrity, and fatty acids in the plasma samples. We compared these plasma concentrations and microbiomes between healthy volunteers and COVID-19 patients, some of whom had unfortunately died by the end of the study enrollment, and performed a correlation analysis between plasma variables and bacterial abundances. Rectal swabs of COVID-19 patients had reduced abundances of several commensal bacteria including Faecalibacterium prausnitzii and an increased abundance of the opportunistic pathogens Eggerthella lenta and Hungatella hathewayi. Furthermore, the oral pathogen Scardovia wiggsiae was more abundant in the oropharyngeal swabs of COVID-19 patients who died. The abundance of both H. hathewayi and S. wiggsiae correlated with circulating inflammatory markers including IL-6, highlighting the possible role of the microbiome in COVID-19 severity and providing potential therapeutic targets for managing COVID-19.IMPORTANCEOutcomes in coronavirus disease 2019 (COVID-19) are highly disparate and are associated with uncontrolled inflammation; however, the individual factors that lead to this uncontrolled inflammation are not fully understood. Here, we report that severe COVID-19 is associated with systemic inflammation, microbial translocation, and microbial dysbiosis. The rectal and oropharyngeal microbiomes of COVID-19 patients were characterized by a decreased abundance of commensal bacteria and an increased abundance of opportunistic pathogens, which positively correlated with markers of inflammation and microbial translocation. These microbial perturbations may, therefore, contribute to disease severity in COVID-19 and highlight the potential for microbiome-based interventions in improving COVID-19 outcomes.

Human ACE2 Gene Replacement Mice Support SARS-CoV-2 Viral Replication and Nonlethal Disease Progression.

Many mouse models of SARS-CoV-2 infection involve expression of the human ACE2 protein, the entry receptor for SARS-CoV-2 Spike protein, in mouse tissues. However, most of these models suffer from nonphysiological regulation of ACE2 expression, which can lead to atypically severe infections and aberrant sites of viral replication. In this report, we developed and characterized an ACE2 gene replacement (ACE2-GR) mouse strain in which the mouse Ace2 genomic locus was replaced by the entire human ACE2 gene locus, and we investigated the ability of these animals to respond to SARS-CoV-2 infection. We show that ACE2-GR mice support SARS-CoV-2 viral replication, but, in stark contrast to the widely used K18-hACE2 transgenic model, this infection leads to a mild disease with no detectable involvement of the CNS. Thus, ACE2-GR mice provide a novel, to our knowledge, model to explore immune responses and long-term consequences of SARS-CoV-2 infection.

Suboptimal activation of antigen-specific CD4+ effector cells enables persistence of M. tuberculosis in vivo.

Adaptive immunity to Mycobacterium tuberculosis controls progressive bacterial growth and disease but does not eradicate infection. Among CD4+ T cells in the lungs of M. tuberculosis-infected mice, we observed that few produced IFN-γ without ex vivo restimulation. Therefore, we hypothesized that one mechanism whereby M. tuberculosis avoids elimination is by limiting activation of CD4+ effector T cells at the site of infection in the lungs. To test this hypothesis, we adoptively transferred Th1-polarized CD4+ effector T cells specific for M. tuberculosis Ag85B peptide 25 (P25TCRTh1 cells), which trafficked to the lungs of infected mice and exhibited antigen-dependent IFN-γ production. During the early phase of infection, ∼10% of P25TCRTh1 cells produced IFN-γ in vivo; this declined to <1% as infection progressed to chronic phase. Bacterial downregulation of fbpB (encoding Ag85B) contributed to the decrease in effector T cell activation in the lungs, as a strain of M. tuberculosis engineered to express fbpB in the chronic phase stimulated P25TCRTh1 effector cells at higher frequencies in vivo, and this resulted in CD4+ T cell-dependent reduction of lung bacterial burdens and prolonged survival of mice. Administration of synthetic peptide 25 alone also increased activation of endogenous antigen-specific effector cells and reduced the bacterial burden in the lungs without apparent host toxicity. These results indicate that CD4+ effector T cells are activated at suboptimal frequencies in tuberculosis, and that increasing effector T cell activation in the lungs by providing one or more epitope peptides may be a successful strategy for TB therapy.

Impaired fitness of Mycobacterium africanum despite secretion of ESAT-6.

BACKGROUND: When compared with Mycobacterium tuberculosis, individuals that live in the same household as an active case of smear-positive pulmonary tuberculosis exposed to M. africanum progress less frequently to active disease within 2 years. A putative ESX-1 secretion apparatus member, Rv3879c, is mutated in M. africanum, and individuals infected with M. africanum less frequently demonstrate T-cell responses to the ESX-1-secreted virulence factor ESAT-6 than those infected with M. tuberculosis. We hypothesized that less frequent progression is caused by impaired secretion of ESAT-6. METHODS: We analyzed in vivo growth and in vitro secretion of ESAT-6 and CFP-10, comparing M. tuberculosis to M. africanum and a strain of M. africanum complemented with M. tuberculosis Rv3879c. RESULTS: ESAT-6 and CFP-10 secretion were similar for all strains, although these were enriched in M. africanum cell lysates, suggesting a modest ESX-1 secretion defect unrelated to the Rv3879c mutation. In mice, M. africanum demonstrated smaller bacterial population sizes than M. tuberculosis but similar numbers and frequencies of ESAT-6-responsive T cells in the lungs. CONCLUSIONS: These results confirm impaired fitness of M. africanum in vivo and indicate that Rv3879c is not required for secretion of ESAT-6 or for its presentation as an antigen to T cells in vivo.

A new hope; the M. tuberculosis strikes back.

The development of new tools to combat TB is counterbalanced by the discovery of previously unknown biological mechanisms used by M. tuberculosis to evade eradication. Two new studies offer both new hope, in a promising ribosome-targeting TB therapy, as well as a new challenge to overcome, in antibiotic resilience.